Summary Three-dimensional electron microscopy (3DEM) is a set of techniques for determining the structures of large proteins and protein complexes. It has been shown that cryogenic EM (cryo-EM) has the ability to determine structures to high enough resolution to unambiguously place side chains in the EM density. Though cryo-EM is increasingly reaching near-atomic resolution, a number of hurdles are present in order to achieve side-chain level resolution structure determination of low-symmetry and no symmetry complexes. Here we propose to develop tool to facilitate high-throughput high-resolution 3DEM. In the first aim, we propose to develop tools to enable automated specimen preparation and screening, and enable a new tools for direct electron detection. In the second aim, we propose to develop tools for tomographic data collection and processing including exploring new modalities for image acquisition and for developing an infrastructure for processing tomographic subvolumes. This research will benefit human health by facilitating the determination of protein structures that are relevant for human disease.